Preserved, non-infectious control cell for use in the identification of a disease through blood testing

ABSTRACT

The invention describes the preparation of non-infectious control cells from normal, non-infectious, non-disease altered blood by depletion or augmentation of one or more cell types found in normal blood to reflect a specific disease state. The control cells so produced are preserved and thereafter reconstituted for use in immunological assays.

RELATED INVENTION

This application is related to U.S. Pat. No. 5,059,518, issued Oct. 22,1991. This application and U.S. Pat. No. 5,059,518 are owned by a commonassignee, Coulter Corporation, Hialeah, Fla.

FIELD OF THE INVENTION

This invention relates to control cells and a method of producing thesame for use in immunoassays. Specifically, this invention relates topreserved non-infectious control cells which have a population or countof at least one specific cell type that is indicative of the populationor count of such cells in the blood or tissue sample of a mammal havinga disease which results in a change in the count of such cells relativeto their count in a normal blood or tissue sample. In particular, thenon-infectious control cells are produced from normal, non-infectious,non-disease altered blood by the depletion or the augmentation of one ormore cell types found in normal blood to reflect a specific diseasestate. The normal blood samples so depleted or augmented are thenpreserved, for example, by lyophilization, so as to have an extendedstorage life for subsequent application in clinical and immunologicalanalytical procedures.

BACKGROUND OF THE INVENTION

Control cells are essential for the accuracy and precision of clinicaland immunological assays. They are needed to insure the reliability andaccuracy of test equipment and methods and to insure reproducibilitythrough time and from laboratory to laboratory. State and federalregulations which govern such assays often require the use ofmulti-level controls in order to demonstrate that equipment isperforming properly over a range of values. In immunological assays,fresh normal cells have been the standard control cells for suchequipment testing. In order to avoid the cost and expense of obtainingand maintaining fresh cells, various methods of preserving fresh cellshave been evaluated. For example, U.S. Pat. No. 5,059,518 (the '518patent) describes the use of lyophilized normal mammalian cells for useas control cells.

Abnormal blood cell samples have also been used as controls to confirmthe presence of a disease or determine its stage, but their use has beenrestricted to fresh or fresh-frozen samples drawn from diseasedpatients. The supply of such samples is thus inherently restricted.Furthermore, since many of such blood samples are associated withinfectious diseases, they are not amenable, for health and social aswell as sometimes technical reasons, to large scale production anddistribution, and they require special handling procedures. Thisinvention describes an alternative to the use of such abnormal controlcells through the use of normal control cell samples which have beenmodified to reflect the population or count of one or more specific celltypes as present in the blood of a person evidencing a specific disease.

SUMMARY OF THE INVENTION

The invention provides for control cells which reflect a specific cellpopulation in a blood sample of a mammal having a disease whichmanifests itself, among other ways, as a change in the type, number orphysiochemical properties of cells present in blood, and a method ofproducing and preserving such control cells. The control cells reflectthe abnormal extant state of a blood cell population arising from: (1)an increase or decrease in the number of cells of a specific type due tothe presence of a disease, the increase or decrease being relative tothe number of such cells in a normal blood sample; or (2) the presenceof cells not normally found in a normal blood samples, such cells beingdifferent in size, shape or other physical characteristics, or havingthereon molecules and/or antigentic sites not found on the normal cells.The control cells of the first type are prepared by depleting a bloodsample of specific cells normally present in blood to less than normallevels or by adding such cells above the normal level present in blood,and subsequently preserving such samples. The control cells of thesecond type are prepared by the addition of cells not normally presentin blood to a normal blood sample and subsequently preserving suchsample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (a)-(d) represents a flow cytometric analysis of a normal bloodsample in which the leukocytes have been conjugated with a PE(phycoerythrin) labelled anti-CD19 monoclonal antibody and a FITC(fluoroscein isothiocyanate) labelled anti-CD10 monoclonal antibody.

FIGS. 2 (a)-(d) represent a flow cytometric analysis of normal bloodsample containing added CD10 positive cells.

FIGS. 3 (a)-(d) represent a flow cytometric analysis of a blood samplefrom a patient having common acute lymphoblastic leukemia.

FIGS. 4 (a)-(d) represents a flow cytometric analysis of lymphocytesfrom a normal blood sample.

FIGS. 5 (a)-(d) represents a cytometric analysis of lymphocytes a bloodsample from an AIDS patient.

FIGS. 6 (a)-(d) represents a cytometric analysis of lymphocytes fromwhich CD4 cells have been depleted according to the invention.

FIG. 7 graphically depicts the percentage decrease in CD4 cells as anormal cell sample is diluted with increasing volumes of a CD4 depletedsample.

FIG. 8 graphically depicts the change in CD4 cell count per cubicmilliliter sample volume as a normal cell sample is diluted withincreasing volumes of a CD4 depleted sample.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "abnormal control cell sample" refers to a cellsample in which the population or count of cells, either total or aspecific type, differs from that of a normal blood sample; or there ispresent in the abnormal blood sample cells which are not present in anormal blood sample. For example, normal values for the total leukocyte(white blood cell or WBC) count range from 4,300 to 10,800 per cu. mm.The normal values for the various cell types in the differentialleukocyte count, are: segmented neutrophils=34-75%, bandneutrophils=0-8%, lymphocytes=12-50%, monocytes=3-15%, eosinophils=0-5%and basophils=0-3% (The Merck Manual, 14th Ed., R. Berkow, ed (MerckSharp & Dohme, Rahway, N.J. 1982), page 2182). In an abnormal bloodsample, these values are different. For example, (1) in a sample from apatient suffering from chronic granulocytic leukemia, the WBC count maybe elevated to range between 15,000 to 500,000 or high (ibid., page1142); (2) a hemophiliac will have a mild to severe deficiency of factorVIII, which is identified as an antigen (ibid, pages 1127-1129); and (3)in a sample from a patient suffering from common acute lymphoblasticleukemia (CALL), cells will have a CD10 antigen not normally presentwhich may be identified specifically by an anti-CD10 monoclonal antibodysuch as J5 (Coulter Corporation Hialeah, Fla).

The term "physiochemical properties" includes the antigeniccharacteristic of cells whether such characteristics are normallypresent or absent as the result of a disease.

The invention may be satisfied by embodiments in many different formsand in relation to different mammalian species. While the detailedexamples described herein relate to humans and to analyses relating toAIDS and to CALL, they are to be considered exemplary of the principlesof the invention and are not intended to limit the scope of theinvention to such specific embodiments. Furthermore, while many of themolecular structures used to identify a type of cell are unique tohumans and do not normally occur in other mammals, practice of theinvention is not restricted to human control cell samples and a methodof

preparing the same. For example, control cell samples could be preparedfor diagnosing feline leukemia virus or simian T-cell lymphotropic virus1 (STLV-I), among others.

Abnormal control cells are usually associated with one of twoconditions. In the first, there is an increase or a decrease in thepopulation or count of a particular type of cells. For example, bloodsamples periodically taken from a HIV (AIDS) infected person will show adecrease in CD4 positive lymphocytes as the disease progresses. In thesecond, there appear cells that are not found in a normal blood sample.For example, the appearance of CD10 positive cells in the peripheralblood samples of patients suffering from Common Acute LymphoblasticLeukemia [CALL; K. A. Foon et al., "Immunologic Classification ofLeukemia and Lymphoma", Blood 68: 1-31 (1986)]. The immunologicalanalysis of a peripheral blood sample for the various cell populationsis performed, usually instrumentally, by associating the binding ofspecific monoclonal antibodies to specific molecular structures on or ina specific type of cell. For example, CD4 positive lymphocytes areidentified by the binding of anti-CD4 monoclonal antibodies to anantigenic site or sites of a CD4 molecule. Several different molecularstructures may have to be identified on a single cell in order toidentify a particular type of cell or a particular type of cellassociated with a specific disease. A broad class of cells may beincreasingly divided into smaller and more specific subsets. In order todistinguish one broad class of cells within several broad classes and tofurther distinguish subsets of the selected broad class, differentlabels may be attached to the different monoclonal antibodies. Forexample, one may use, alone or in combination, radioactive elementcontaining molecules, enzymes and dyes, especially fluorescent dyes, assuch labels. Fluorescent dyes are preferred and examples of such dyesinclude fluorescein, fluorescein isothiocyanate (FITC),tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE or RD),phycoerythrin-Texas Red conjugates and allophycocyanin, among others.

In general, the method of preparing the control cells for which adecreased number of specific cells is important, comprises poolingnormal, leukocyte rich, anti-coagulated blood samples, lysing the redblood cells (RBCs) therein and washing the samples to remove lysisdebris or removing RBCs by any other suitable procedure, for example, asdescribed in U.S. Pat. No. 4,752,863, removing a specific cell type fromthe sample by use of a monoclonal antibody conjugated to a separablesubstrate such as glass, ceramic or polymeric beads, preferably magneticbeads, and adding back the removed cells to specific levels which may beindicative of various stages of the progression of a disease. Thesamples are then preserved for long term storage, for example, bylyophilizing the sample according to the procedures described in U.S.Pat. No. 5,059,518, whose teachings are incorporated herein by referenceor by other suitable cell preservation techniques. The control cellsthus produced have present a normal spectrum of leukocyte cells exceptfor those specific, disease related cells whose number has beenincreased or decreased, or those specific disease related cells whichhave been added.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are given by way of illustration and are not tobe taken as limiting the invention.

Patients having HIV exhibit decreased levels of CD4 (T4) cells in theirblood. Since CD4 cells are important in combatting many of the secondarydiseases which directly cause the death of a AIDS patient, assaying theCD4 population is indicative of the progress of the HIV infection andthe ability of the patient to stave off secondary infections. Whileanalysis of a patient's blood sample is absolutely necessary, it isdesirous that the control samples used in the analysis be non-infectiousso as to minimize accidental transmission of any disease.

EXAMPLE 1 Preparation of CD4 Depleted Normal Blood Cells for Use as AIDSControl Cells

A number of ACD (acid citrated dextrose) or CPD (citrated, phosphateddextrose) anticoagulated, leukocyte rich, normal blood packs arecombined in a vessel to give a bulk leukocyte rich blood sample. The redblood cells in the sample are lysed using a 0.83 wt% ammonium chloridesolution. After lysis, the leukocyte cells are washed a plurality oftimes with Hepes-saline-bovine serum albumin (HSB) solution. Theleukocyte cells are then separated from the final wash and placed in avessel with an isotonic 10% trehalose solution as a preservative medium.A portion of the leukocyte cells are reserved for later use. Separately,an anti-CD4 monoclonal antibody such as T4 (Coulter Corporation,Hialeah) is conjugated to magnetic beads by methods such as thosedescribed in U.S. Pat. No. 4 752 563 among others. The anti-CD4conjugated magnetic beads are added to the washed leukocyte sample andmixed for about 30 minutes. The beads and cells attached thereto arethen removed from the vessel using a magnetic device; for example, ahand held magnetic or a commercially available magnetic separator. Thecells remaining in the vessel are centrifuged, the supernatant liquid isremoved and the cells resuspended in an isotonic trehalose solution,preferably an isotonic 10% trehalose solution, as preservation medium.Samples containing different numbers of CD4 cells are then prepared bymixing the CD4-depleted sample with varying quantities of the reservedleukocyte cells or normal, red blood cell free leukocyte cells fromother samples with known number of CD4 cells. For example:

Level 1 CD4 cells: Equal volumes of the reserved leukocytes and the CD4depleted leukocytes are mixed to yield a sample having 50% of the CD4cells present in the original normal pooled blood sample.

Level 2 CD4 cells: Three volumes of the reserved leukocytes and onevolume of CD4 depleted leukocytes are mixed to yield a sample having 25%of the CD4 cells present in the original pooled blood sample.

Level 3 CD4 cells: Nine volumes of the reserved leukocytes and onevolume of CD4 depleted leukocytes are mixed to yield a sample having 10%of the CD4 cells present in the original pooled blood sample.

FIGS. 7 and 8 graphically present CD4 control cells prepared asdescribed. FIG. 7 depicts the change in the percent positive cells as anormal CD4 containing sample is diluted with increasing amount of CD4depleted blood. FIG. 8 depicts the change in the absolute number of CD4positive cells per cubic millimeter sample as dilution increases.

Alternatively, additional normal ACD or CPD anti-coagulated,leukocyte-rich blood packs are pooled, the red blood cells are lysed andthe leukocytes are washed as previously described herein. The number ofCD4 cells present in the pooled sample may be determined by flowcytometry using fluorescently labelled T4 monoclonal antibody. Aliquotsof this pooled, CD4 containing sample may then be mixed with varyingamounts of CD4 depleted leukocytes to prepare control cell sampleshaving varying concentrations of CD4 cells.

EXAMPLE 2 Preparation of CD10 (CALLA) Augmented Control Cells

A plurality of normal ACD or PCD anticoagulated, leukocyte-rich redblood packs are pooled and the red blood cells then lysed using an 0.83wt% ammonium chloride solution. After lysis, the leukocyte cells arewashed a plurality of times with HSB. The leukocyte cells are thenseparated from the final wash and placed in a vessel with isotonic 10%trehalose solution as the preservative medium. Cultured CALLA (CD10positive, common acute lymphoblastic leukemia antigen) positive cellsare separately prepared using commercially available CALLA positivecells, an American Type Culture Collection (ATCC) CALLA sample, or cellsfrom other sources. The cells used here are from the ATCC, depositnumber CRL 1596. The cultured CD10+ cells are then added in differentamounts to aliquots of normal leukocyte cells in the preservativesolution.

EXAMPLE 3 Lyophilization of Stabilized Normal Cells for Use asNon-infectious Diseased Cell Controls

The control cells prepared as described in Examples 1 and 2 arelyophilized in accordance with U.S. Pat. No. 5,059,518 whose teachingsare incorporated herein by references. Specifically the Level 1, 2 or 3CD4 control cells or CALLA control cells suspended in the isotonic 10%trehalose solution are placed in 300 μL lyophilization vials, the vialscapped and cooled to 4° C., and then agitated to assure even and smoothdispersion of cells before the vials are placed in a freezer at about-70° C. for at least one hour. After the freezing time, the vials areimmediately placed in a lyophilizer for a period of about 15 hours. Thelyophilized vials are removed from the lyophilizer after the expirationof the fifteen hour cycle and stored at refrigeration temperatures ofbetween 2°-8° C. The control cells may be stored for six months or more.The lyophilized cells may be reconstituted by filling the lyophilizationvial to its 300 μL volume with deionized or distilled water. Forconducting a control assay on a flow cytometer, the resuspendedreconstituted cells are stained, or otherwise labelled or marked, andthereafter analyzed by standard flow cytometer procedures. Theresuspended cells can also be used in other types of assays or othersuitable diagnostic protocols.

FIGS. 1, 2 and 3, (a)-(d) compare normal cells (FIG. 1) with patientcells (FIG. 3) and control cells prepared according to the invention(FIG. 2). These figures indicate that the assay is able, using thecontrol cells, to detect the abnormal CD10+ and CD19+ cells which wouldbe present in a diseased patient blood sample.

FIGS. 4, 5 and 6, (a)-(d), indicate that the assay is able to detect thelow number of CD4+ cells that would be seen in an AIDS patient. In FIG.4, Quad 1 represents CD4+ cells as present in a normal blood sample. InFIG. 5, Quad 1 represents the depleted number of CD4+ cells as seen inan AIDS patient. In FIG. 6, Quad 1 shows that the equipment is able todetect the depleted CD4+ as represented by the depleted control cells ofthe invention.

We claim:
 1. Control cells comprising leukocyte cells which have been lyophilized with an isotonic trehalose solution, said control cells reflecting the type, number and physiochemical properties of leukocyte cells in the blood of a mammal having a disease, which disease has as one of its manifestations a change in the type, number and physiochemical properties of the leukocyte cells in the blood of said diseased mammal, said control cells further identified as being normal mammalian leukocyte cells in which:(a) at least one specific subset of normal leukocyte cells has been artificially depleted to a count below that present in a normal leukocyte cell sample, or (b) at least one specific subset of normal leukocyte cells which has been artificially increased to a count above that present in a normal leukocyte cell sample, or (c) at least one specific subset of cells, not present in a normal leukocyte cell sample, has been added to said normal leukocyte sample,in order that the control cells which result from the lyophilization of (a), (b) or (c) have a leukocyte cells distribution similar to that of the diseased mammal.
 2. The control cells according to claim 1 in reconstituted form wherein said cells are useful as biological controls in immunological assays.
 3. Control cells in accordance with claim 1 in which said cells are derived from normal human peripheral blood cells. 